Dynamics of Internalization and Recycling of the Pro-Metastatic Membrane Type 4-Matrix Metalloproteinase (MT4-MMP) in Breast Cancer Cells

MT4-MMP (MMP17) is a glycosylphosphatidyl inositol (GPI)-anchored membrane-type MMP expressed on the cell surface of human breast cancer cells. In triple negative breast cancer cells, MT4-MMP promotes primary tumor growth and lung metastases. Although trafficking and internalization of the transmembrane MT1-MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI-anchored MT4-MMP. Here, we investigated the fate and cellular trafficking of MT4-MMP by analyzing its homophilic complex interactions, internalization and recycling dynamics compared to an inert form, MT4-MMP-E249A. Oligomeric and dimeric complexes were analyzed by co-transfection of cells with FLAG- or Myc-tagged MT4-MMP by reducing and non-reducing immunoblots and co-immunoprecipitation experiments. The trafficking of MT4-MMP was studied using an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and Western blot analysis. We demonstrate that MT4-MMP forms homophilic complexes at the cell surface, internalizes in early endosomes, and some of the enzyme is either auto-degraded or recycled to the cell surface. Our data indicate that MT4-MMP is internalized by the CLIC/GEEC pathway, a mechanism that differs from other MT-MMP members. Although MT4-MMP localizes with caveolin-1, MT4-MMP internalization was not affected by inhibitors of caveolin-1 or clathrin endocytosis pathways but was reduced by cdc42 or RhoA silencing with siRNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4-MMP, which may have implications in the design of novel therapeutic strategies for metastatic breast cancer. This article is protected by copyright. All rights reserved

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c

Editor's Note: EGFR Activation and Signaling in Cancer Cells Are Enhanced by the Membrane-Bound Metalloprotease MT4-MMP.

peer reviewedThe editors are publishing this note to alert readers to concerns about this article (1). In Fig. 2, the actin loading control bands for cyclin D1 and cyclin D2 are identical-the authors clarified that the Western blots for cyclin D1 and cyclin D2 were performed on the same samples, however, this was not indicated in the figure legend. Additionally, in Fig. 6C, the p-EGFR bands in MDA-MB-231 cells showing stimulation by TGFa treatment are identical to the p-EGFR bands showing stimulation by EGF treatment. In the original submission of this manuscript, a correct version of this figure was used to show both TGFa and EGF could stimulate p-EGFR in control vector (CTR)- and MT4-MMP-expressing (MT4) MDA-MB-231 cells, but these panels were mistakenly duplicated in the revised and final versions of the manuscript

Implication de la MT4-MMP dans la progression tumorale des cancers du sein triple-négatifs

La MT4-MMP appartient à une grande famille d’endopeptidases matricielles dont le rôle dans la progression tumorale a largement été décrit dans la littérature. Parmi elle, la MT4-MMP se différencie des autres MMPs par une série de caractéristiques biochimiques qui lui confère des fonctions physiologiques et pathologiques distinctes. Les travaux antérieurs de notre laboratoire ont révélé l’implication de la MT4-MMP dans la tumorigenèse mammaire. Cette enzyme contribue à la croissance de xénogreffes de cellules tumorales triple-négatives en stimulant l’angiogenèse et promeut la dissémination hématogène des cellules tumorales aux poumons. Ce travail de thèse a mis en lumière une action directe de la MT4-MMP sur la croissance tumorale par la stimulation de la prolifération cellulaire. Cet effet mitogène est promu par la transactivation de l’EGFR. La régulation de la MT4-MMP à la surface cellulaire est importante dans l’activation du récepteur tyrosine kinase. Nous avons démontré que la MT4-MMP est internalisée par une voie d’endocytose particulière (CLIC/GEEC) et qu’elle est majoritairement recyclée à la surface cellulaire où elle forme des dimères et oligomères. Dans une étude clinique rétrospective menée sur des patientes avec un TNBC, nous avons observé que plus de 70% des patientes exprimaient à la fois la MT4-MMP et l’EGFR. Nous avons également identifié la MT4-MMP comme un indicateur prédictif de la réponse des patientes à la chimiothérapie et l’erlotinib, un anti-EGFR. En absence de traitements ciblés, les TNBC restent associés à un mauvais pronostic. La surexpression de l’EGFR dans plus de la moitié des TNBC en fait une cible thérapeutique de choix. Les nombreuses études cliniques menées à ce jour avec des drogues anti-EGFR, ont été effectuées sur des patientes avec un TNBC non sélectionnées et ont été décevantes. L’analyse immunohistochimique de la MT4-MMP pourrait être utilisée pour sélectionner les patientes susceptibles de répondre à l’erlotinib. Notre travail ouvre également de nouvelles perspectives dans l’élaboration d’inhibiteurs thérapeutiques de la MT4-MMP comme des anticorps neutralisants

MT4-MMP: The GPI-Anchored Membrane-Type Matrix Metalloprotease with Multiple Functions in Diseases

MT4-MMP (or MMP17) belongs to the Membrane-Type Matrix Metalloproteinase (MT-MMP) family. This family of proteases contributes to extracellular matrix remodeling during several physiological processes, including embryogenesis, organogenesis, tissue regeneration, angiogenesis, wound healing, and inflammation. MT4-MMP (MMP17) presents unique characteristics compared to other members of the family in terms of sequence homology, substrate specificity, and internalization mode, suggesting distinct physiological and pathological functions. While the physiological functions of MT4-MMP are poorly understood, it has been involved in different pathological processes such as arthritis, cardiovascular disease, and cancer progression. The mt4-mmp transcript has been detected in a large diversity of cancers. The contribution of MT4-MMP to tumor development has been further investigated in gastric cancer, colon cancer, head and neck cancer, and more deeply in breast cancer. Given its contribution to different pathologies, particularly cancers, MT4-MMP represents an interesting therapeutic target. In this review, we examine its biological and structural properties, and we propose an overview of its physiological and pathological functions

A new approach to the isolation of TSH receptor proteins

info:eu-repo/semantics/publishe